PROJECT SUMMARY Ca2+ regulates a range of activities in hepatocytes, from secretion to metabolism to proliferation. This single second messenger can regulate these different cell functions in part because it is differentially regulated in the various microdomains within the cell. For example, secretion is regulated by Ca2+ in the apical region of the cytoplasm, lipid metabolism is regulated by Ca2+ in mitochondria, and liver regeneration is regulated by Ca2+ within the nucleoplasm. The inositol 1,4,5-trisphosphate receptor (InsP3R) is the only intracellular Ca2+ release channel in hepatocytes, and so it is largely responsible for establishing these various microdomains and for the resulting effects. There are three InsP3R isoforms, only two of which are expressed in hepatocytes under normal conditions. The type 1 InsP3R (InsP3R-1) accounts for 20% of InsP3Rs in hepatocytes. It is distributed throughout the cytoplasm and our recent evidence furthermore suggests it preferentially couples ER to mitochondria and is responsible for mitochondrial Ca2+ signaling. The type 2 InsP3R (InsP3R-2) normally accounts for 80% of InsP3Rs in hepatocytes. It is concentrated in a specialized ER region beneath the canalicular membrane and regulates insertion and activation of transporters important for bile secretion. The type 3 InsP3R (InsP3R-3) normally is present in negligible amounts in hepatocytes, but our preliminary data suggest that its expression increases dramatically in patients with chronic hepatocellular diseases, such as alcoholic hepatitis and hepatitis C viral infection. Our preliminary findings further suggest that the subcellular distribution of InsP3R-3 is distinct from that of InsP3R-1 or 2. Therefore, the hypothesis of this project is that inflammation increases InsP3R-3 expression in hepatocytes and this is responsible for the impaired liver regeneration that occurs in chronic liver disease and cirrhosis. We will test this hypothesis through the following specific aims: (1) Determine the factors that regulate InsP3R-3 expression in hepatocytes. We will determine the role of specific transcription factors, miRNAs, and gene methylation in InsP3R-3 expression under normal circumstances and in specific disease states. (2) Identify the factors that determine the patterns of subcellular targeting of InsP3R-3 in hepatocytes and its effect on Ca2+ signals. We will identify the targeting signals that determine the subcellular distribution of InsP3R-3 in hepatocytes, and determine the subcellular regions in which InsP3R-3 increases Ca2+. (3) Determine the effects of InsP3R-3 expression in hepatocytes on cell proliferation, apoptosis and liver regeneration. We will examine disease conditions in which hepatocyte InsP3R-3 expression is increased and liver regeneration is impaired, and determine whether and how InsP3R- 3 participates in the impaired liver regeneration. Collectively, these studies will provide new insights about the basic biological mechanisms by which subcellular processes are regulated and control cell function, while simultaneously establishing how these subcellular events are important for the pathogenesis of liver disease.